Adeno-associated virus (AAV) is a parvovirus having a single-stranded DNA genome of about 4.6 kb. Unlike other viruses, AAV is naturally defective, requiring coinfection with a helper virus (e.g. adenovirus or herpes virus) to establish a productive infection. No human disease has been found to be associated with AAV infection (Blacklow et al., 1968). The host range of AAV is broad; unlike retroviruses, AAV can infect both quiescent and dividing cells in vitro and in vivo (Flotte et al., 1993; Kaplitt et al., 1994; Podsakoff et al., 1994; Russell et al., 1994) as well as cells originating from different species and tissue types in vitro (Lebkowski et al., 1988; McLaughlin et al., 1988). When infection occurs in the absence of a helper virus, wild-type AAV can integrate into the cellular genome as a provirus, until it is rescued by superinfection with adenovirus. (Handa et al., 1977; Cheung et al., 1980; Laughlin et al., 1986).
The AAV genome is relatively simple, containing two open reading frames (ORFs) flanked by short inverted terminal repeats (ITRs). The ITRs contain, inter alia, cis-acting sequences required for virus replication, rescue, packaging and integration. The integration function of the ITR permits the AAV genome to integrate into a cellular chromosome after infection.
The nonstructural or replication (Rep) and the capsid (Cap) proteins are encoded by the 5xe2x80x2 and 3xe2x80x2 ORFs, respectively. Four related proteins are expressed from the rep gene; Rep78 and Rep68 are transcribed from the p5 promoter while a downstream promoter, p19, directs the expression of Rep52 and Rep40. The larger Rep proteins (Rep78/68) are directly involved in AAV replication as well as regulation of viral gene expression (for review, see Muzyczka, 1992). The cap gene is transcribed from a third viral promoter, p40. The capsid is composed of three proteins of overlapping sequence; the smallest (VP-3) is the most abundant. Because the inverted terminal repeats are the only AAV sequences required in cis for replication, packaging, and integration (Samulski et al., 1989), most AAV vectors dispense with the viral genes encoding the Rep and Cap proteins and contain only the foreign gene inserted between the terminal repeats.
Interest in AAV as a vector in gene therapy results from several unique features of its biology. Stable genetic transformation, ideal for many of the goals of gene therapy, may be achieved by use of such AAV vectors. Furthermore, the site of integration for AAV is well-established as being on chromosome 19 of humans. This predictability removes the danger of random insertional events into the cellular genome that may activate or inactivate host genes or interrupt coding sequences, consequences that limit the use of vectors whose integration is random, e.g., retroviruses. Because the rep protein mediates the integration of AAV, it is believed that removal of this protein in the construction of AAV vectors result in altered integration patterns. In addition, AAV has not been associated with human disease, obviating many of the concerns that have been raised with virus-derived gene therapy vectors.
Notwithstanding the attractive aspects of AAV-based vectors, rapid progress in their evaluation for gene therapy has been hampered by the inability to produce recombinant viral stocks at large-scale and to high titer. The conventional method for production of recombinant AAV (rAAV) vectors is cotransfection of one plasmid containing the vector and a second helper plasmid encoding the AAV Rep and Cap proteins into 293 cells infected with adenovirus (e.g. Lebkowski et al., 1988; Samulski et al., 1989, Muzyczka, N., 1992, Kaplitt et al., 1994; Einerhand et al., 1995). This method is cumbersome and results in a low yield of rAAV, typically 104-105 infectious or transducing units/ml. Strategies to improve this scheme have included increasing transfection efficiency by complexing plasmid DNA to adenoviral particles via polylysine (Mamounas et al., 1995), delivering the vector sequences as part of a recombinant adenovirus (Thrasher et al., 1995) and amplification of helper plasmid copy number by linkage to a SV40 replicon (Chiorini et al. 1995).
Progress in the development of AAV as a gene therapy vector has been limited by an inability to produce high titer recombinant AAV stock using the approaches described above. The limitations to date have been thought to derive from inadequate production of the AAV proteins required in trans for replication and packaging of the recombinant AAV genome. Trans-based strategies to vector production are those that modulate the level of proteins required in trans to effectuate AAV vector production. Attempts to increase the levels of these proteins have included placing the AAV rep gene under the control of the HIV LTR promoter (Flotte, F. R. et al., Gene Therapy 2:29-37, 1995) to increase protein levels and the development of cell lines that express the rep proteins (Yang, Q. et al., J. Virol. 68: 4847-4856, 1994).
The limitations in producing high titer AAV vector stock may also result from a failure to include AAV cis-required elements in the recombinant AAV vector design. Cis-based strategies to increase vector production are those that provide DNA sequences required in cis (in tandem) with the recombinant DNA to be packaged into the AAV vector particle. The trans and cis functions are related. Trans-required proteins are necessary to effectuate vector production, but they require cis-acting sequences in the recombinant AAV genome in order to be functionally active. Therefore, high yield AAV vector production requires a coordinated strategy of trans-based and cis-based improvements so that progress in the development of AAV as a standard gene therapy vehicle may be realized.
Thus, there is a need in the art for methods and compositions which enable production of high titer recombinant AAV (rAAV) preparations that are free from wild-type AAV and Adenovirus helper contamination.
The present invention is directed to methods for generating high titer, contaminant free, recombinant AAV vectors.
The present invention provides methods and genetic constructs for producing AAV recombinant vectors conveniently and in large quantities.
The present invention further provides methods for the delivery of all essential viral proteins required in trans for high yields of recombinant AAV.
The present invention provides recombinant AAV vectors for use in gene therapy, using trans- and cis-based strategies.
The present invention also provides novel packaging cell lines which obviate the need for cotransfection of vector and helper plasmids.
The invention is also directed to helper plasmids and vector plasmid backbone constructs that are used in these methods.
The present invention-provides a reporter assay for determining AAV vector yield.
Further provided are recombinant AAV vectors in a pharmaceutically acceptable carrier.
The present invention also provides methods of delivering a transgene of interest to a cell.
Compositions and methods for delivering a DNA sequence encoding a desired protein to a cell are provided by the present invention.
Still further provided are transgenic non-human mammals that express a human chromosome 19 AAV integration locus.